_ This program project application comprises a team of investigators with complimentary research strengths and resources that will be harnessed for the development of novel small molecules that antagonize the action of the HIV-1 vif protein. Project 2 (Virologic support for SAR and mechanisms of inhibitor resistance) will predominantly support drug discovery/SAR efforts in Project 1 as well as identifying mutations conferring inhibitor resistance in vitro and in vivo. Specific responsibilities of Project 2 include: Evaluation of antiviral activity of lead compounds and their analogs in permissive and non-permissive cells. The antiviral activities of analogs emerging from the SAR exercise of Project 1 will be compared in permissive cells (vif-independent replication) and non-permissive cells (vif-dependent replication) and will be used to drive the design of analogs with more potent antiviral activity. Evaluation of antiviral activity in primary cells relevant to CNS reservoirs of viral replication. Macrophage are the principle cellular targets in the CNS and infected macrophage drive the neuropathogenic manifestations of viral replication. Therefore, vif antagonists will be evaluated for antiviral activity in primary macrophage in vitro. Identification of mechanisms governing inhibitor resistance in vitro and in vivo. HIV-1 will be passaged in non-permissive cells in increasing concentrations of prioritized Vif analogs in order to derive inhibitor resistant virus. In addition, plasma viral RNA from inhibitor-treated macaques (Project 3) will be cloned and sequenced to identify mutations conferring resistance in vivo. Impact of vif-inhibitor resistance on sensitivity to other ARV classes will be conducted with Core B. The virologic studies outlined in Project 2 will guide the SAR studies and drive the prioritization of analogs that go forward for analysis of in vivo efficacy in macaques.